Increasing butanol titers and selectivity in Clostridium acetobutylicum Ryan Sillers To increase the butanol titers and selectivity in Clostridium acetobutylicum we replaced the promoter of the alcohol/aldehyde dehydrogenase (aad) gene with the phosphotranbutyrylase (ptb) promoter and combined this with CoAT downregulation to minimize acetone production. This led to early production of high alcohol (butanol plus ethanol) titers and overall solvent titers of 30 g/L. To increase then the carbon

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... ux towards butanol, we examined thiolase (thl) overexpression. The combined thl overexpression with aad overexpression decreased, as expected, acetate and ethanol production while increasing acetone and butyrate formation. thl overexpression in strains with CoAT downregulation did not significantly alter product formation thus suggesting that a more complex metabolic engineering strategy is necessary to achieve improved butanol titers and selectivity. The aad with the ptb promoter was also used in the M5 strain and restored butanol production to wildtype levels. Thiolase overexpression was combined with aad overexpression aiming to enhance butanol formation. While acetate was reduced, butanol titers were not improved. We generated acetate kinase (AK) and butyrate kinase (BK) knockout (KO) mutants of M5, reducing the respective acid formation. We could not successfully transform the BKKO M5 strain. The AKKO M5 strain overexpressing aad produced less acetate, but also less butanol compared to the M5 aad overexpression strain. The difficulty in generating high butanol producers without acetone and acid production is likely PDC AAD, AdhE2 AAD, AdhE2,